MicroLab - Quiz

Control of Microbial Growth, Microbiome, and Biochemical Tests (Experiment 7)

aseptic techniques

Semmelweiss and Lister helped develop this to prevent contaminations of surgical wounds in 1800

before the development of aseptic techniques

• nosocomial infections caused death in 10% of surgeries

• up to 25% of mothers that delivered in hospitals died due to infections

early civilization practices for controlling microbial growth

• salting

• smoking

• pickling

• drying

• exposure of food and clothing to sunlight

• some use spices in cooking to mask the taste of spoiled food 

  • some spices prevent spoilage

sterilization

killing or removing all forms of microbial life (including endospores) in a material or an object

the most commonly used method of sterilization

heating

commercial sterilziation

• heat treatment that kills endospores of Clostridium botulinum which is the causative agent of botulism in canned food

• does not kill endospores of thermophiles, which are not pathogens and may grow at temperatures above 45C

disinfection

• reducing the number of pathogenic microorganisms to the point where they no longer cause diseases

• usually involves the removal of vegetative or non-endospore forming pathogens

• may use physical or chemical methods

disinfectant

applied to inanimate object

antiseptic

applied to living tissues (antisepsis)

degerming

mechanical removal of most microbes in a limited area

• example: alcohol swab on the skin

sanitization

use of chemical agents on food-handling equipment to meet public health standards and minimize chances of disease transmission

• example: hot soap and water

sepsis

• comes from the Greek word for decay or putrid

• indicates bacterial contamination

asepsis

absence of significant contamination

aseptic technique definition

• used to prevent contamination of surgical instruments, medical personnel, and patients during surgery

• also used to prevent bacterial contamination in the food industry

bacteriostatic agent

an agent that inhibits the growth of bacteria, but does not necessarily kill them. 

suffix: -statis

to stop or steady

germicide

an agent that kills certain microorganisms

bactericide

kills bacteria; most do not kill endospores

viricide

kills or inactivates viruses

fungicide

kills fungi

sporicide

kills bacterial endospores and fungal spores 

rate of microbial death

• when the bacterial populations are heated or treated antimicrobial chemicals, they usually die at a constant rate

factors that influence the effectiveness of antimicrobial treatment

1. number of microbes = the more microbes present, the more time it takes to eliminate a population 

2. type of microbe = endospores are very difficult to destroy. vegetative pathogens vary widely on susceptibility to different methods of microbial control

3. environmental influences = presence of organic material (blood, feces, saliva) tends to inhibit antimicrobials, pH, etc.

4. time of exposure = chemical antimicrobials and radiation treatments are more effective at longer times. in heat treatments, longer exposure compensates for lower temperatures 

heat

• kills microorganisms by denaturing their enzymes and other proteins

• heat resistance varies widely among microbes

thermal death point (IDP)

lowest temperature at which all of the microbes in a liquid suspension will be killed in ten minutes

thermal death time (TDT) 

minimal length of time in which all bacteria will be killed at a given temperature

decimal reduction time (DRT)

time in minutes at which 90% of bacteria at a given temperature will be killed; used in the canning industry

moist heat

• kills microorganisms by coagulating their proteins

• much more effective than dry heat

• reliable sterilization with most heat required temperatures above that of boiling water

boiling

• heat to 100C or more at sea level

• hills vegetative forms of bacterial pathogens, almost all viruses, and fungi and their spores within 10 minutes or less.

• endospores and some viruses are not destroyed this quickly. however, brief boiling will kill most pathogens

Hepatitis virus

can survive up to 30 minutes of boiling

endospores

can survive up to 20 hours or more of boiling

autoclave

• chamber which is filled with hot steam under pressure

• preferred method of sterilization, unless material is damaged by heat, moisture, or high pressure

  • temperature of the steam reaches 121C at twice the atmospheric pressure

  • most effective when organisms contact steam directly or are contained in a small volume of liquid 

    • all organisms and endospores are killed within 15 minutes

  • require more time to reach the center of solid or large volume of liquid

pasteurization

• developed by Louis Pasteur to prevent spoilage of beverages

• used to reduce microbes responsible for the spoilage of beer, milk, wine, juices, etc.

Classic method of pasteurization

milk was exposed to 65C for 30 minutes

high temperature short time pasteurization (HTST)

used today, the milk is exposed to 72C for 15 seconds

ultra high temperature pasteurization (UHT)

milk is treated at 140C for 3 seconds then cooled very quickly in a vacuum chamber

• advantage: milk ca be stored at room temperature for several months

dry heat

kills by oxidation effects

direct flaming

used to sterilize inoculating loops and needles; heat metal until it has a red glow

incineration

effective way to sterilize disposable items (paper cups, dressings) and biological wastes

hot air sterilziation

• place objects in an oven

• requires 2 hours at 170C for sterilization

• dry heat transfers heat less effectively to a cool body than moist heat

filtration

• removal of microbes by passage of a liquid or gas through a screen-like material with small pores.

• used to sterilize heat sensitive materials like vaccines, enzymes, antibiotics, and some culture media

high efficiency particulate air filters (HEPA)

used in operating rooms and burn units to remove bacteria from the air

membrane filters

• uniform pore size

• used in the industry of research

different sizes of membrane filters

• 0,22 and 0.45 um pores = used to filter most bacteria; does not retain spirochetes, mycoplasmas, and viruses

• 0.01 um pores = retains all viruses and some large proteins

low temperature

effect depends on the microbe and treatment applied

refrigeration

• temperatures from 0-7C

• bacteriostatic effects

• reduces metabolic rate of most microbes so they cannot reproduce or produce toxins

freezing

• temperatures below 0C

• over a third of vegetative bacteria may survive 1 year

• most parasites are killed by a few days of freezing

flash freezing

does not kill most microbes

slow freeszing

more harmful because ice crystals disrupt cell structure

dessication

• in the absence of water, microbes cannot grow or reproduce, but some remain viable for years

• after water becomes available, they start growing again

susceptibility to dessication varies widely

• Neisseria gonorrhea = only survives about one hour

• Mycobacterium tuberculosis = may survive several months; viruses are fairly resistant

• Clostridium spp. and Bacillus spp. = may survive decades

osmotic pressure

the use of high concentrations of salts and sugars in food is used to increase the osmotic pressure and create a hypertonic environment

• yeasts and molds = more resistant to high osmotic pressure

• Staphylococci spp. that live on skin = fairly resistant to high osmotic pressure

plasmolysis

• as water leaves the cell, plasma membranes shrink away from the cell wall

• cells may not die, but usually stops growing

ionizing radiation

• gamma rays, x rays, electron beams, or higher energy rays

• have short wavelengths (< 1 nanometer)

• dislodges electrons from atoms and form ions

• causes mutations in DNA and produce peroxides

• used to sterilize pharmaceuticals and disposable medical supplies

• disadvantages: penetrates human tissues; may cause genetic mutations in humans

ultraviolet light (non-ionizing radiation)

• wavelength is longer than 1 nanometer

• damages DNA by producing thymine dimers, which causes mutations

• used to disinfect operating rooms, nurseries, and cafeterias

• disadvantages: damages skin, eyes; doesn’t penetrate paper, glass, or cloth

microwave radiation

• wavelength ranges from 1 millimeter to 1 meter

• heat is absorbed by water molecules

• may kill vegetative cells in moist food

• bacterial endospores, which does not contain water, are not damaged by microwave radiation

• solid food are unevenly penetrated by microwaves

• trichinosis outbreaks have been associated with port cooked in microwaves

types of disinfectants

1. phenols and phenolics

2. halogens

3. alcohols

4. heavy metals

5. quaternary ammonium compounds (quats)

6. aldehydes 

7. gaseous sterilizers

8. peroxygens

phenol

• carbolic acid

• was first used by Lister as a disinfectant

• rarely used today because it is a skin irritant and has a strong odor

• used in some throat sprays and lozenges 

• acts as a local anesthetic

phenolics

• chemical derivatives of phenol

cresols

derived from coal tar (lysol)

bisphenols (pHisoHex)

• effective against gram-positive staphylococci and streptococci

• used in nurseries

• excessive use in infants may cause neurological damage

advantages of phenols and phenolics

• destroy plasma membranes and denature proteins

• stable

• persist for longer times after application 

• remains active in the presence of organic compounds

halogens

effective alone or in compounds

iodine

• tincture of iodine = alcohol solution; the first antiseptics used

• combines with amino acid tyrosine in proteins and denatures proteins

• stains the skin and clothes

• somewhat irritating

iodophors 

• compounds with iodine that are slow releasing

• takes several minutes to act

• used as skin antiseptic in surgery

• not effective against bacterial endospores

• examples: betadine, isodine

alcohol

• kills bacteria, fungi, but not endospores or naked viruses

• acts by denaturing proteins and disrupting cell membranes

• evaporate, leaving no residue

• used to mechanically wipe microbes off skin before injections or blood drawing

• not good for open wounds, because it causes proteins to coagulate

ethanol

• drinking alcohol

• optimum concentration is 70%

isopropanol

• rubbing alcohol

• better disinfectant than ethanol; cheaper and less volatile

heavy metals

• includes copper, selenium, mercury, silver, and sic

• oligodynamic action = very tiny amounts are effective

silver

1% silver nitrate used to protect infants against gonorrheal eye infections until recently

mercury

organic mercury compounds like Merthiolate and mercurochrome are used to disinfect skin wounds

copper

copper sulfate is used to kill algae in pools and fish tanks

selenium

• kills fungi on their spores

• used for fungal infections

• also used in dandruff shampoos

zinc

• zinc chloride = mouthwashes

• zinc oxide = antifungal agents in paint

quaternary ammonium compounds (quats)

• widely used surface active agents

• cationic (positively charge) detergents

• effective against gram—positive bacteria, less effective against gram-negative bacteria

• can also destroy fungi, amoeba, and enveloped viruses

• Zephiran, cepacol = also found in our lab spray bottles

• Pseudomonas = strains that are resistant and can grow in the presence of quats are the big concern of hospitals 

• advantages: strong antimicrobial action, colorless, odorless, tasteless, stable, nontoxic

• disadvantages: forms foam, organic matter interferes with effectiveness, neutralized by soap and anionic detergents

aldehydes

• includes some of the most effective microbials

• inactivates proteins by forming covalent crosslinks with several functional groups

formaldehyde gas

• excellent disinfectants

• commonly used as formalin, a 37% aqueous solution

• formalin was used extensively to preserve biological specimens and inactivate viruses and bacteria in vaccines

• irritates mucous membranes, and has a strong odor

• also used in mortuaries for embalming

• formaldehyde inactivates microorganisms by alkylating the amino and sulfhydryl groups of proteins and ring nitrogen atoms of purine bases.

glutaraldehyde

• less irritating and more effective than formaldehyde

• one of the few chemical disinfectants that is a sterilizing agent

• a 2% solution of glutaraldehyde (Cidex) 

  • bactericidal, tuberculocidal, and viricidal in 10 minutes

  • sporicidal in 3 to 10 hours

• commonly used to disinfect hospital instruments

• also used in mortuaries for embalming 

gaseous sterilizers

• chemicals that sterilize in a chamber similar to an autoclave

• denatures proteins by replacing functional groups with alkyl groups

ethylene oxide

• kills all microbes and endospores, but requires exposure of 4-18 hours

• toxic and explosive in pure form

• highly penetrating

• most hospitals have ethylene oxide chambers to sterilize mattresses and large equipment

peroxygens (oxidizing agents)

• oxidize cellular components of treated microbes

• disrupts membranes and proteins

ozone

• used along with chlorine to disinfect water

• helps neutralize unpleasant tastes and odors

• more effective killing agent than chlorine, but less stable and more expensive

• highly reactive form of oxygen

• made by exposing oxygen to electricity or UV light

hydrogen peroxide

• used as an antiseptic

• not good for open wounds because it can be quickly broken down by catalase that is present in human cells

• effective in disinfection or inanimate objects

• sporicidal at higher temperatures

• used by the food industry and to disinfect contact lenses

benzoyl peroxide

used in acne medications

paracetic acid

• one of the most effective liquid sporicides available

• sterilant:

  • kills bacteria and fungi in less than 5 minutes

  • kills endospores and viruses within 30 minutes

• used widely in disinfection of food and medical instruments because it does not leave toxic residues

microbiome or normal flora

• microorganisms that are frequently found in a particular site in a normal, healthy individual

• some are found in association with humans/animals only. majority are bacteria

• symbolic relationship with the host

• subject to constant changes

• altered by antimicrobial agents 

types of normal flora

1. commensals

2. residents

3. transients

4. carrier state

commensals

natural relationship with the host

residents

present for invariable periods

transients

establish itself briefly, excluded by host defenses or competition from residents

carrier state

• potentially pathogenic

• examples: 

  • Streptococcus pneumoniae

  • Neisseria meningitidis in the throat of a healthy individual 

origin of normal flors

• newborn sterile in utero

• after birth, exposed to flora of mother’s genital tract, skin, respiratory tract flora of those handling them, and organisms in the environment

beneficial effects of normal flora

1. immunostimulation = antibody development

2. exclusionary effect (vacuum effect) = protection from external invaders

3. production of essential nutrients = vitamin K and B by some normal intestinal flora

   1. example: E.coli

immunostimulation

The gut microbiota (commensals) profoundly affects the host immune system and is a critical area of scientific and clinical importance. It is now evident that the gut flora influences autoimmune diseases both inside and outside the gut , where environmental factors like antibiotic overuse can increase the risk of these diseases through microbiota-mediated immunomodulation.

normal flora - may be a source of opportunistic infections

In patients with impaired defense mechanisms. eg Staphylococcus epidermidis, Escherichia coli

normal flora - some may cross react with normal tissue components

antibodies to various ABO group arise because of cross reaction between intestinal flora and the antigens of A &B blood substances.

normal flora - production of carcinogens

Some normal flora may modify through their enzymes chemicals in our diets into carcinogens.

• example: artificial sweeteners may be enzymatically modified into bladder carcinogens

distribution of normal flora

• internal organs (except alimentary tract) are sterile at health

• local maintained by:

  • local defense mechanisms

  • chemical substances in serum and tissues

    • example: complement antibodies

  • phagocytic activity or polymorphmonomnucleocytes (PMN)

areas of the body with normal flors

• GIT: mouth and large colon

• urogenital tract = vagina, distal 1/3 of the urethra 

• skin

distribution and frequency of bacteria in the intestine